The major goals of the research proposed in this application are (a) to develop convenient methods for isolating and culturing large numbers of hormonally-responsive rabbit and rat renal cortical collecting tubule (CCT) cells; (b) to grow rabbit CCT cells with asymmetry on Millipore filters and to determine if there is a sidedness to the release of prostaglandin E2 (PGE2) formed in response to arginine vasopressin (AVP) and bradykinin; (c) to determine if prostaglandin endoperoxide (PGH) synthase is located on the plasma membrane (in addition to the endoplasmic reticulum and nuclear membrane) of collecting tubule epithelia and other prostaglandin-forming renal cells; and (d) to determine if the PGH synthase associated with the nucleus is on the inner or outer membrane of the nuclear envelope. We have recently prepared a monoclonal antibody specific for an antigen on the surface of rabbit and rat collecting tubule epithelia. We propose to isolate rabbit and rat CCT cells by immunoadsorption. We will then develop culture conditions which best support retention of differentiated hormonal responses. This methodology should be of general use for studies of collecting tubule biochemistry. We will use rabbit CCT cells grown with asymmetry on Millipore filters to determine if PGE2 is released exclusively to the basolateral surface. A sidedness to release is anticipated based on results from other laboratories indicating the PGE2 acts only from the basolateral surface of the collecting tubule to inhibit both Na+ resorption and the hydroosmotic effect of AVP. The subcellular location of PGH synthase will be determined by immunoelectron microscopy using rabbit antiserum specific for the enzyme. It is unclear whether prostaglandin formation can occur on the plasma membrane as well as the endoplasmic reticulum and nuclear membrane of prostaglandin-forming cells. The capacity of cells to form prostaglandins at several intracellular locations would have important implications for the regulation of prostaglandin synthesis. A related, as yet unresolved question, is whether prostaglandins are formed on the inner or outer surface of the nuclear envelope. If synthesis can occur on the inner membrane of the envelope, the results would imply that prostaglandins play a role in the regulation of nuclear events.